Project Summary/Abstract Copy number variants (CNVs) ? increases and decreases in the number of copies of genomic regions ? are a pervasive class of genetic variation that contribute to rapid adaptive evolution and human phenotypic variation and disease. Moreover, CNVs are the first step in processes that result in genome evolution such as gene family expansion and the generation of novel functions through modification of duplicate genes. Despite the importance of CNVs in human health and evolution, many fundamental questions about their role in evolving populations and their functional consequences remain unsolved. Experimental microbial evolution in chemostats is an ideal system for studying the evolutionary constraints and functional consequences of CNVs. To gain quantitative insights into the role of CNVs in evolving populations, we have developed a novel CNV reporter system using a constitutively expressed fluorescent protein gene linked to a locus at which CNVs are known to be repeatedly generated and selected. By coupling this system to a lineage tracking system using random molecular barcodes we have developed a powerful novel framework for studying the diversity and dynamics of CNVs in complex populations. We will use this novel system to address three fundamental questions. In aim 1, we will address the dynamics with which CNVs are selected in fluctuating environments. Using a combination of two-color CNV reporters and lineage tracking we will investigate the dynamics and diversity of CNV selection in variable chemostat environments. In aim 2, we will identify the determinants of CNV fitness effects. We will establish a collection of hundreds of unique CNV alleles that are molecularly characterized using whole genome sequencing and uniquely marked by DNA barcodes. We will use pooled fitness assays in a range of environmental conditions to quantify the fitness of each lineage and use these data to identify determinants of condition dependent and independent fitness fitness costs and benefits of CNVs. In aim 3, we will test the effect of CNVs on variability of gene expression and phenotypes. Using single cell RNA sequencing we will test whether CNVs results in increased variation in expression levels of both the genes contained within a CNV and all genes throughout the genome. We will also test whether CNVs result in increased variability in phenotypes using a high throughput microcolony growth rate assay. Our study will make use of the unparalleled efficiency and rigor afforded by the budding yeast model system and the unprecedented resolution provided by our new method for CNV detection to address fundamental questions of widespread significance for our understanding of CNVs in evolution and disease. As CNVs are universal in evolution and disease findings from our study will have a high impact on the field of genomics, evolution, and human health.